-Previously, we developed a highly sensitive assay for viremia, capable of detecting a single copy of HIV-1 RNA in plasma. This sensitivity, capable of quantitating virions in plasma down to 0.3 copies RNA/ml or less, represents a 150-fold improvement over previous ultra-sensitive assays. In collaboration with colleagues at Abbott Laboratories, we have found that viremia in patients is independent of regimen, but strongly associated with pretherapy virus levels, implying that, even after 7 years of treatment, cells infected prior to therapy survive to make more virus. In collaboration with R. Siliciano, we completed a clinical trial demonstrating the lack of additional suppression of HIV-1 viremia on intensification of standard antiretroviral therapy with either protease inhibitor or NNRTI intensification. These studies are consistent with our recent findings indicating little or no active cycles of HIV-1 replication during standard drug therapy. During the past year, we extended these studies in an additional intensification trial with the new integrase inhibitor, raltegravir. In collaboration with D. McMahon, we demonstrated no evidence of additional suppression by raltegravir intensification. In collaboration with ACTG investigators (R. Gandhi, PI), we participated in a large multisite, randomized study of raltegravir intensification using a longer duration of intensification. HVIB funds and personnel were employed to complete single copy assays and maintain database sand participate in analysis of data. This study confirmed our pilot study and demonstrated that prolonged intensification did not result in additional decline in viremia. Taken together these experiments have provided strong evidence that the source of persistent viremia during antiretroviral suppression is not active cycles of HIV replication in short lived CD4 T cells. -These natural history and interventional studies indicate that new approaches are necessary to eradicate HIV-1 infection. Several basic and clinical lines of evidence have suggested that the cytokine interferon alpha may have substantial effects on HIV replication. In collaboration with S. Kottilil, we conducted a small retrospective review of stored samples we noted decreased levels of HIV RNA in HIV-HCV co-infected individuals receiving interferon as part of HCV therapy. We have developed a new protocol, currently under IRB review, to investigate the effect of interferon alpha in suppressing persistent viremia in patients suppressed on standard antiretroviral therapy (collaboration with D. McMahon). In addition we are collaborating with E. Reid to measure effects of the proteasome inhibitor, bortezomib, on persistent HIV-1 viremia in infected individuals with suppressed viemia who are receiving bortezomib as therapy for co-morbid lymphoma (Bench to Bedside, 2008). These studies will shed light on potential new directions designed to eradicate HIV-1 infection in individuals suppressed by antiretroviral therapy. -We have initiated new studies to investigate the characteristics of low level viremia. We have initiated new analyses to investigate the establishment of low level viremia. In collaboration with M. DiMascio, we are investigating immune correlates of first and second phase decay kinetics in a cohort of well characterized patients followed at NIH for prolonged period ( greater than 8 y, NIH protocol 97-I-0082, now 08-I-0221;F. Maldarelli, PI). In initial analyses, we have identified significant variability in phase II decay kinetics and are investigating immune correlates (CD4, CD8, CD45RO/RA cell numbers) responsible for variability. In addition, with M. King, we are investigating immune correlates of persistent low level viremia in individuals suppressed for prolonged periods. In initial studies, we found that patients in 08-I-0221 who have been suppressed for greater than 8 years still have small but statistically significant increase in immune activation when compared to uninfected individuals. We are investigating whether the degree of immune activation is correlated with level of persistent viremia. -During the past several years, a number of independent line of evidence have suggested a strong contribution of gut associated lymphoid tissue (GALT) to HIV replication. Defects in gut function as a result of HIV infection result in increased translocation of bacterial components into blood, a process which has been proposed to drive a chronic inflammatory state, increase T cell activation, and virus production from chronically infected cells. In order to investigate the role of bacterial translocation in persistent viremia we are investigating the effects of reductions in bowel flora in HIV persistent viremia. Specifically we are investigating the effects of the non-absorbable antibiotic, rifaxamin, on bacterial translocation, immune activation, and persistent viremia. Rifaximin is an FDA approved antibacterial agent that is highly effective in reducing bacterial flora in the gastrointestinal tract. Our hypothesis is that rifaximin-induced reductions in bacteria in the gastrointestinal tract will result in reductions in bacterial translocation from GALT into the bloodstream, and thereby reduce both immune activation and persistent viremia. We received a new Bench to Bedside Award (2009) for this project, and will be collaborating with D. McMahon and A. Ganesan. -The single copy analytic technique is also being utilized as a benchmark for the development of new approaches to measure HIV-1. With I. Hewlett and L. Demeter, we are participating in development of europium based p24 detection assays (Bench to Bedside 2008). -The same single copy assay is being used (in collaboration with B. Walker) to probe the levels of viremia in the rare HIV-1-infected patients who are able to control their viremia at very low levels in the absence of therapy, as well as in a well-described NIH cohort of patients with HLA-restricted HIV-1 replication (with S. Migueles, M. Conners, and H.C. Lane) and also (in collaboration with D. Margolis) to see whether a drug that activates HIV expression (i.e., Suberoylanilide hydroxamic acid, SAHA) can affect the level of persistent virus. -We are expanding the utility of the single copy assay to to quantitate cell associated HIV nucleic acid. With J. Mellors, we are developing techniques to measure both HIV RNA and DNA, including new assays to measure circular forms of HIV DNA. These assays will be useful in investigating the course of HIV replication both in peripheral blood derived cells as well as tissue derived material. -We are investigating the nature of HIV-1 replication during suppressive therapy in specific patient populations. We have initiated new collaborations with J. Ananworanich and I. Sereti to investigate HIV-1 subtype A/E viremia in patients suppressed on antiretroviral therapy who initiated therapy during acute seroconversion. -[Corresponds to Project 1 in the April 2007 site visit report of the Host-Virus Interaction Branch, HIV Drug Resistance Program]